Glycohydrolytic enzymes are a class of enzymes which have been employed in various diagnostic applications, such as for measuring PMN phagocytic activity and periodontal disease activity, for classifying bacteria, and as a component of conjugates in enzyme immunoassay procedures. Conventionally, the determination of glycohydrolytic enzymes, particularly .beta.-glucuronidase, involves the use of a hydrophobic substrate. Such substrates include synthetic compounds such as phenolphthalein glucuronide and methylumbelliferyl glucuronide, and metabolic products such as the glucuronides of steroids and bilirubin. However, the affinity of hydrolytic enzymes, such as .beta.-glucuronidase, for highly polar substrates has not been described.
Wolfbeis, et al. (Analytical Biochemistry, Vol. 129, 365-370, 1983), describe the synthesis of esters of acetic, butyric, caprylic and oleic acids with 1-hydroxypyren-3,6,8-trisulfonic acid of the formula [I]: ##STR1## wherein R is --CH.sub.3, --(CH.sub.2).sub.2 --CH.sub.3, --CH.sub.2).sub.6 --CH.sub.3 and --(CH.sub.2).sub.7 --CH.dbd.CH--(CH.sub.2).sub.7 --CH.sub.3, respectively. Although such esters show violet fluorescence (.lambda..sub.max 390 and 408 nanometers) when excited below 385 nanometers, they do not fluoresce when excited at 460 nanometers. However, hydrolysis of such esters results in the formation of an anion of the formula [II]: ##STR2## which exhibit both longwave excitation and emission maxima and a large Stokes' shift of 60 nanometers and have been employed as substrates for determining several hydrolases.
Fishman, et al. ("Naphthol AS-BI .beta.-D-Glucosiduronic Acid, its Synthesis and Suitability as a Substrate for .beta.-Glucuronidase", J. Histochem, and Cytochem., Vol. 12, 298-305, 1964), describe the synthesis of naphtol AS-BI .beta.-D-glucosiduronic acid of the formula [III]: ##STR3## Fishman, et al., also describe the chemical and physical properties of the compound of the formula [III], as well as its behavior as a substrate for .beta.-D-glucuronidase.
Bollenback, et al. (J. Am. Chem. Soc., 77, 3310-3315, 1955), describe the synthesis of methyl(tri-O-acetyl-(.alpha. and .beta.)-D-glucopyranosyl bromide)-uronates of the formula [IV] below from glucouronolactone, as well as the synthesis of various methyl(aryl-tri-O-acetyl-(.alpha. and .beta.)-D-glucopyranosid)-uronates of the formula [V] below from the methyl(tri-O-acetyl-(.alpha. and .beta.)-D-glucopyranosyl bromide)-uronates: ##STR4## The methyl(aryl-tri-O-acetyl-(H.sup.T)-D-glucopyranosid)-uronates described by Bollenback, et al., include those where R in the formula [V] is phenyl, 1-naphthyl and 2-naphthyl, and the alpha anomers of the compounds of the formula [IV] and [V] where R is o-chlorophenyl, p-chlorophenyl, o-bromophenyl, p-bromophenyl, o-iodophenyl, o-tolyl, m-tolyl, p-tolyl, o-hydroxyphenyl, 1-naphthyl, 2-naphthyl, methyl gentisate and o-nitrophenyl are also described.